There are various bleeding disorders caused by deficiencies of blood coagulation factors. The most common disorders are hemophilia A and B, resulting from deficiencies of blood coagulation factor VIII and IX, respectively. Another known bleeding disorder is von Willebrand's disease.
In plasma FVIII exists mostly as a noncovalent complex with VWF and its coagulant function is to accelerate factor IXa dependent conversion of factor X to Xa Due to the complex formation of FVIII and VWF it was assumed for a long time that FVIII and VWF functions are two functions of the same molecule. Only in the seventies it became clear that FVIII and VWF are separate molecules that form a complex under physiologic conditions. In the eighties then the dissociation constant of about 0.2 nmol/L was determined (Leyte et al., Biochem J 1989, 257: 679-683) and the DNA sequence of both molecules was studied.
Classic hemophilia or hemophilia A is an inherited bleeding disorder. It results from a chromosome X-linked deficiency of blood coagulation FVIII, and affects almost exclusively males with an incidence of between one and two individuals per 10,000. The X-chromosome defect is transmitted by female carriers who are not themselves hemophiliacs. The clinical manifestation of hemophilia A is an increased bleeding tendency. Before treatment with FVIII concentrates was introduced the mean life span for a person with severe hemophilia was less than 20 years. The use of concentrates of FVIII from plasma has considerably improved the situation for the hemophilia A patients increasing the mean life span extensively, giving most of them the possibility to live a more or less normal life. However, there have been certain problems with the plasma derived concentrates and their use, the most serious of which have been the transmission of viruses. So far, viruses causing hepatitis B, non-A non-B hepatitis and AIDS have hit the population seriously. Since then different virus inactivation methods and new highly purified FVIII concentrates have recently been developed which established a very high safety standard also for plasma derived FVIII.
The cloning of the cDNA for FVIII (Wood et al. 1984. Nature 312:330-336; Vehar et al. 1984. Nature 312:337-342) made it possible to express FVIII recombinantly leading to the development of several recombinant FVIII products, which were approved by the regulatory authorities between 1992 and 2003. The fact that the central B domain of the FVIII polypeptide chain residing between amino acids Arg-740 and Glu-1649 does not seem to be necessary for full biological activity has also led to the development of a B domain deleted FVIII.
The mature FVIII molecule consists of 2332 amino acids which can be grouped into three homologous A domains, two homologous C domains and a B Domain which are arranged in the order: A1-A2-B-A3-C1-C2. The complete amino acid sequence of mature human FVIII is shown in SEQ ID NO:15. During its secretion into plasma FVIII is processed intracellularly into a series of metal-ion linked heterodimers as single chain FVIII is cleaved at the B-A3 boundary and at different sites within the B-domain. This processing leads to heterogeneous heavy chain molecules consisting of the A1, the A2 and various parts of the B-domain which have a molecular size ranging from 90 kDa to 200 kDa. The heavy chains are bound via a metal ion to the light chains, which consist of the A3, the C1 and the C2 domain (Saenko et al. 2002. Vox Sang. 83:89-96). In plasma this heterodimeric FVIII binds with high affinity to von Willebrand Factor (VWF), which protects it from premature catabolism. The half-life of non-activated FVIII bound to VWF is about 12 hours in plasma.
Coagulation FVIII is activated via proteolytic cleavage by FXa and thrombin at amino acids Arg372 and Arg740 within the heavy chain and at Arg1689 in the light chain resulting in the release of von Willebrand Factor and generating the activated FVIII heterotrimer which will form the tenase complex on phospholipid surfaces with FIXa and FX provided that Ca2+ is present. The heterotrimer consists of the A1 domain, a 50 kDa fragment, the A2 domain, a 43 kDa fragment and the light chain (A3-C1-C2), a 73 kDa fragment. Thus the active form of FVIII (FVIIIa) consists of an A1-subunit associated through the divalent metal ion linkage to a thrombin-cleaved A3-C1-C2 light chain and a free A2 subunit relatively loosely associated with the A1 and the A3 domain.
To avoid excessive coagulation, FVIIIa must be inactivated soon after activation. The inactivation of FVIIIa via activated Protein C (APC) by cleavage at Arg336 and Arg562 is not considered to be the major rate-limiting step. It is rather the dissociation of the non covalently attached A2 subunit from the heterotrimer which is thought to be the rate limiting step in FVIIIa inactivation after thrombin activation (Fay et al. 1991. J. Biol. Chem. 266 8957, Fay & Smudzin 1992. J. Biol. Chem. 267:13246-50). This is a rapid process, which explains the short half-life of FVIIIa in plasma, which is only 2.1 minutes (Saenko et al. 2002. Vox Sang. 83:89-96).
In severe hemophilia A patients undergoing prophylactic treatment FVIII has to be administered intravenously (i.v.) about 3 times per week due to the short plasma half-life of FVIII of about 12 to 14 hours. Each i.v. administration is cumbersome, associated with pain and entails the risk of an infection especially as this is mostly done at home by the patients themselves or by the parents of children being diagnosed for hemophilia A.
It would thus be highly desirable to create a FVIII with increased functional half-life allowing the manufacturing of pharmaceutical compositions containing FVIII, which have to be administered less frequently.
Several attempts have been made to prolong the half-life of non-activated FVIII either by reducing its interaction with cellular receptors (WO 03/093313A2, WO 02/060951A2), by covalently attaching polymers to FVIII (WO 94/15625, WO 97/11957 and U.S. Pat. No. 4,970,300), by encapsulation of FVIII (WO 99/55306), by introduction of novel metal binding sites (WO 97/03193), by covalently attaching the A2 domain to the A3 domain either by peptidic (WO 97/40145 and WO 03/087355) or disulfide linkage (WO 02/103024A2) or by covalently attaching the A1 domain to the A2 domain (WO2006/108590).
Another approach to enhance the functional half-life of FVIII or VWF is by PEGylation of FVIII (WO 2007/126808, WO 2006/053299, WO 2004/075923) or by PEGylation of VWF (WO 2006/071801) which pegylated VWF by having an increased half-life would indirectly also enhance the half-life of FVIII present in plasma.
As none of the above described approaches has yet resulted in an approved FVIII pharmaceutical and as introducing mutations into the FVIII wild-type sequence or introducing chemical modifications entails at least a theoretical risk of creating immunogenic FVIII variants there is an ongoing need to develop modified coagulation factor VIII molecules which exhibit prolonged half-life.
In view of a potential thrombogenic risk it is more desirable to prolong the half-life of the non-activated form of FVIII than that of FVIIIa.
VWF, which is missing, functionally defect or only available in reduced quantity in different forms of von Willebrand disease (VWD), is a multimeric adhesive glycoprotein present in the plasma of mammals, which has multiple physiological functions. During primary hemostasis VWF acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix such as collagen. Moreover, VWF serves as a carrier and stabilizing protein for procoagulant FVIII. VWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The amino acid sequence and the cDNA sequence of wild-type VWF are disclosed in Collins et al. 1987, Proc Natl. Acad. Sci. USA 84:4393-4397. The precursor polypeptide, pre-pro-VWF, consists of a 22-residue signal peptide, a 741-residue pro-peptide and the 2050-residue polypeptide found in mature plasma VWF (Fischer et al., FEBS Lett. 351: 345-348, 1994). After cleavage of the signal peptide in the endoplasmatic reticulum a C-terminal disulfide bridge is formed between two monomers of VWF. During further transport through the secretory pathway 12 N-linked and 10 O-linked carbohydrate side chains are added. More important, VWF dimers are multimerized via N-terminal disulfide bridges and the propeptide of 741 amino acids length is cleaved off by the enzyme PACE/furin in the late Golgi apparatus. The propeptide as well as the high-molecular-weight multimers of VWF (VWF-HMWM) are stored in the Weibel-Pallade bodies of endothelial cells or in the α-Granules of platelets.
Once secreted into plasma the protease ADAMTS13 cleaves VWF within the A1 domain of VWF. Plasma VWF therefore consists of a whole range of multimers ranging from single dimers of 500 kDa to multimers consisting of up to more than 20 dimers of a molecular weight of over 10,000 kDa. The VWF-HMWM hereby having the strongest hemostatic activity, which can be measured in ristocetin cofactor activity (VWF:RCo). The higher the ratio of VWF:RCo/VWF antigen, the higher the relative amount of high molecular weight multimers.
Defects in VWF are causal to von Willebrand disease (VWD), which is characterized by a more or less pronounced bleeding phenotype. VWD type 3 is the most severe form in which VWF is completely missing, VWD type 1 relates to a quantitative loss of VWF and its phenotype can be very mild. VWD type 2 relates to qualitative defects of VWF and can be as severe as VWD type 3. VWD type 2 has many sub forms some of them being associated with the loss or the decrease of high molecular weight multimers. Von VWD type 2a is characterized by a loss of both intermediate and large multimers. VWD type 2B is characterized by a loss of highest-molecular-weight multimers.
VWD is the most frequent inherited bleeding disorder in humans and can be treated by replacement therapy with concentrates containing VWF of plasmatic or recombinant origin. VWF can be prepared from human plasma as for example described in EP 05503991. EP 0784632 describes a method for isolating recombinant VWF.
In plasma FVIII binds with high affinity to von VWF, which protects it from premature catabolism and thus, plays in addition to its role in primary hemostasis a crucial role to regulate plasma levels of FVIII and as a consequence is also a central factor to control secondary hemostasis. The half-life of non-activated FVIII bound to VWF is about 12 to 14 hours in plasma. In von Willebrand disease type 3, where no or almost no VWF is present, the half-life of FVIII is only about 6 hours, leading to symptoms of mild to moderate hemophilia A in such patients due to decreased concentrations of FVIII. The stabilizing effect of VWF on FVIII has also been used to aid recombinant expression of FVIII in CHO cells (Kaufman et al. 1989, Mol Cell Biol).
Until today the standard treatment of Hemophilia A and VWD involves frequent intravenous infusions of preparations of FVIII and VWF concentrates or of concentrates comprising a complex of FVIII and VWF derived from the plasmas of human donors or in case of FVIII that of pharmaceutical preparations based on recombinant FVIII. While these replacement therapies are generally effective, e.g. in severe hemophilia A patients undergoing prophylactic treatment FVIII has to be administered intravenously (i.v.) about 3 times per week due to the short plasma half life of FVIII of about 12 hours. Already above levels of 1% of the FVIII activity in non-hemophiliacs, e.g. by a raise of FVIII levels by 0.01 U/ml, severe hemophilia A is turned into moderate hemophilia A. In prophylactic therapy dosing regimes are designed such that the trough levels of FVIII activity do not fall below levels of 2-3% of the FVIII activity in non-hemophiliacs. Each i.v. administration is cumbersome, associated with pain and entails the risk of an infection especially as this is mostly done in home treatment by the patients themselves or by the parents of children being diagnosed for hemophilia A. In addition the frequent i.v. injections inevitably result in scar formation, interfering with future infusions. As prophylactic treatment in severe hemophilia is started early in life, with children often being less than 2 years old, it is even more difficult to inject FVIII 3 times per week into the veins of such small patients. For a limited period, implantation of port systems may offer an alternative. Despite the fact that repeated infections may occur and ports can cause inconvenience during physical exercise, they are nevertheless typically considered as favorable as compared to intravenous injections.
The in vivo half-life of human VWF in the human circulation is approximately 12 to 20 hours. In prophylactic treatment of VWD e.g. of type 3 it would also be highly desirable to find ways to prolong the functional half-life of VWF.
Another approach to enhance the functional half-life of VWF is by PEGylation (WO 2006/071801) which pegylated VWF by having an increased half-life would indirectly also enhance the half-life of FVIII present in plasma.
However the chemical conjugation of PEG or other molecules to therapeutic proteins always entails the risk, that the specific activity is reduced due to shielding of important interaction sites with other proteins, chemical conjugation adds an additional step in the manufacture of such proteins decreasing final yields and making manufacture more expensive. Also the long term effects on human health are not known as currently known PEGylated therapeutic proteins do not need to be administrated lifelong as it would be the case for a VWF to be administered in prophylaxis of von Willebrand disease or in for a FVIII to be administered in hemophilia A.
It would thus be highly desirable to obtain a long-lived VWF which is not chemically modified.
In the prior art fusions of coagulation factors to albumin (WO 01/79271), alpha-fetoprotein (WO 2005/024044) and immunoglobulin (WO 2004/101740) as half-life enhancing polypeptides have been described. These were taught to be attached to the carboxy- or the amino-terminus or to both termini of the respective therapeutic protein moiety, occasionally linked by peptidic linkers, preferably by linkers consisting of glycine and serine.
Ballance et al. (WO 01/79271) described N- or C-terminal fusion polypeptides of a multitude of different therapeutic polypeptides fused to human serum albumin. Long lists of potential fusion partners are described without disclosing experimental data for almost any of these polypeptides whether or not the respective albumin fusion proteins actually retain biological activity and have improved properties. Among said list of therapeutic polypeptides also FVIII and VWF are mentioned.
A C-terminal fusion would not have been seriously considered by the man skilled in the art as the C2 domain of FVIII at the very C-terminal part of FVIII between amino acid 2303 and 2332 of FVIII comprises a platelet membrane binding site which is essential for FVIII function. This is why there are many amino acid mutations known in this region which lead to hemophilia A. It was thus surprising that a relatively large heterologous polypeptide like albumin can be fused to the C-terminal part of FVIII without preventing FVIII function by preventing platelet binding. In addition, the C2 domain also contains a binding site for VWF. This site together with the amino acid sequence 1649-1689 is responsible for the high affinity binding of FVIII to VWF. Therefore, a man skilled in the art would also not have expected that a FVIII with a C-terminal albumin fusion would retain its binding to VWF.
It was surprisingly found that in contrast to the prediction by Ballance et al. an albumin fusion to the N-terminus of FVIII was not secreted into the culture medium. Therefore and because of the reasons detailed above it was now even more surprisingly found that a FVIII fused at its C-terminal part to albumin is secreted into the culture medium and retains its biological function including binding to membranes of activated platelets and to VWF.
It was also surprising to find that the modified FVIII of the invention shows an increase of in vivo recovery by about 20% compared to the wild type FVIII.
A man skilled in the art would also not have considered fusing human albumin to the N- or the C-terminus of VWF. In an N-terminal fusion the albumin part would be cleaved off during propeptide processing. Or if the propeptide would be omitted the multimerization would not take place. As detailed above the C-terminus of VWF is essential for the initial dimerization and secretion as shown by Schneppenheim et al. (Schneppenheim R. et al. 1996. Defective dimerization of VWF subunits due to a Cys to Arg mutation in VWD type IID. Proc Natl Acad Sci USA 93:3581-3586; Schneppenheim R. et al. 2001. Expression and characterization of VWF dimerization defects in different types of VWD. Blood 97:2059-2066.), Baronciani et al. (Baronciani L. et al. 2000. Molecular characterization of a multiethnic group of 21 patients with VWD type 3. Thromb. Haemost 84:536-540), Enayat et al. (Enayat M S et al. 2001. Aberrant dimerization of VWF as the result of mutations in the carboxy-terminal region: identification of 3 mutations in members of 3 different families with type 2A (phenotype IID) VWD. Blood 98:674-680) and Tjernberg et al. 2006. Homozygous C2362F VWF induces intracellular retention of mutant VWF resulting in autosomal recessive severe VWD. Br J Haematol. 133:409-418). Therefore the man skilled in the art would not consider fusing a large protein like human albumin to the C- or N-terminus of VWF as he would expect that normal dimerization or multimerization of VWF would be impaired. As the higher multimers of VWF are the ones most active in primary hemostasis the man skilled in the art would have looked for other ways to prolong the functional half-life of VWF.
It was now surprisingly found that fusing heterologous polypeptides such as albumin to the C-terminal part of VWF, not only permits expression and secretion of VWF chimeric proteins from mammalian cells but also results in modified VWF molecules that retain significant VWF activity and form high molecular weight multimers. In addition, such modified VWF molecules exhibit prolonged in vivo half-life and/or improved in vivo recovery.